Acyltransferase

ABSTRACT

The invention relates to at least one nucleotide sequence, derived from a nucleotide sequence encoding an acyltransferase polypeptide comprising at least one membrane-spanning region, encoding an improved active membrane independent acyltransferase polypeptide in which at least one amino acid residue of the membrane-spanning region has been deleted and/or substituted as compared to the original acyltransferase polypeptide, wherein the encoded active membrane independent acyltransferase polypeptide can produce fatty acid esters and/or fatty acid thioesters such as triacylglycerols, diacylglycerols, monoacylglycerols, phospholipids, glycolipids, waxesters, acylated carbohydrates, acylated amino acids, and lysolipids, e.g. lysophosphospholipid, lysolecithin. Thereby one single acyltransferase can be used for the production of a huge number of products. The invention also relates to means and methods for the production of such an improved active membrane independent acyltransferase and the use of such a membrane independent acyltransferase in industry.

FIELD OF INVENTION

The invention relates to at least one nucleotide sequence, derived froma nucleotide sequence encoding an acyltransferase polypeptide comprisingat least one membrane-spanning region, encoding an improved activemembrane independent acyltransferase polypeptide in which at least oneamino acid residue of the membrane-spanning region has been deletedand/or substituted as compared to the original acyltransferasepolypeptide, wherein the encoded active membrane independentacyltransferase polypeptide can produce fatty acid esters and/or fattyacid thioesters such as triacylglycerols, diacylglycerols,monoacylglycerols, phospholipids, glycolipids, waxesters, acylatedcarbohydrates, acylated amino acids, and lysolipids, e.g.lysophosphospholipid, lysolecithin. Thereby one single acyltransferasecan be used for the production of a huge number of products. Theinvention also relates to means and methods for the production of suchan improved active membrane independent acyltransferase and the use ofsuch a membrane independent acyltransferase in industry.

BACKGROUND OF INVENTION

A phospholipid: diacylglycerol acyltransferase (PDAT) has biochemicallybeen characterised in yeast and plants and a gene, LRO1, encoding thePDAT enzyme was identified in yeast (Dahlqvist et al., 2000, PNAS97:6487-6492). The enzyme was shown to catalyse the formation oftriacylglycerols (TAG) by an acyltransfer from phospholipids todiacylglycerols (DAG). Furthermore, the enzymatic activity was found tobe localised in the microsomal fraction. The gene encoding the PDATenzyme was shown to have sequence homologies to the lecithin:cholesterol acyltransferase (LCAT) gene family. The LCAT enzyme is usedfor the treatment of LCAT deficiencies, such as arteriosclerosis byincreasing the activity of LCAT in serum of the mammal to a leveleffective to decrease the accumulation of cholesterol (WO9717434). Thediet habit used by large groups of people today result in highcholesterol values with all other problems, which follow.

Lipases are enzymes that are primarily responsible for the hydrolysis ofglycerolipids such as triacylglycerols. However, it is well known thatlipases also under certain conditions in water free systems, cancatalyse interesterification (Gandhi, 1997, J Am Oil Chem Soc 74 (6):621-634). The wide berth for employment in a variety of reactions andbroad substrate specificity has rendered the lipases to be very usefulin a variety of applications such as production of pharmaceuticals,cosmetics, detergents, foods, perfumery, and other organic syntheticmaterials. One example is the use of an immobilised lipase for thesynthesis of waxes (U.S. Pat. No. 4,826,767 and U.S. Pat. No.6,162,623). The low stability, low activity or selectivity encounteredoccasionally with a number of these enzymes have been the chief obstaclehindering a more rapid expansion of industrial lipase technology intonew applications on a large scale.

Additionally, mass-production of waxes have been performed by culturingmicroorganisms, together with fatty-acids, wherein acyltransferasespresent within the microorganism convert the fatty acids into waxesters,such as by using the microorganism Staphylococcus lentus (JP 1320989).Another example is the use of Arthrobacter ceroformans for theproduction of waxesters (Koronelli et al., 1979, Vestn. Mosk. Univ. Ser16, Biol 3:62-64). Other examples are the use of transgenic hostsharbouring a gene encoding an acyltransferase for the production ofwaxes, as described in WO 9310241 and U.S. Pat. No. 5,445,947.

Industrial application using the above mentioned lipases as biocatalyst,for the production of a variety of waxesters, is limited to the group oflipases and the restrictions these enzymes have both regarding theproducts that could be produced and the conditions by which theseenzymes are active. For example, the esterification must occur in waterfree solvents and under reduced pressure.

By the use of microorganisms there are limitations such as the need ofseveral purification steps after the synthesis of the waxesters to beable to remove the microorganism and other impurities, which comes alongwith the culturing method. There are also difficulties in obtaining highyields of the waxesters. The microorganism may be one that naturallyencodes enzymes suitable for the synthesis of waxesters, or agenetically modified microorganism, which by the modification obtainsthe ability to produce waxesters.

Furthermore, the waxesters that can be synthesised today are limited dueto the substrate specificity of the enzymes catalysing the wax estersynthesis in these microorganisms. Moreover, these enzymes are integralmembrane enzymes, which render it impossible to use such enzymes asbiocatalyst in a cell free system such as in an industrial reactor.

There is a need for new improved enzymes, which enables the productionof variety of fatty acid esters to high yields in cost-efficientindustrial processes. Examples of fatty acid esters are structuredglycerol fatty acid esters such as triacylglycerols with a specific acylgroup at the sn2 positions that differs as compared to that of the outerpositions and diacylglycerols with specific acylgroups. Production offat-soluble fatty acid esters by acylation of water-soluble molecules,such as flavours and vitamins, is another example of desirable fattyacid esters. Other valuable fatty acid esters of interest are waxesters(i.e. fatty acids esterified to long chain alcohols), or fatty acidesters of molecules such as carbohydrates and amino acids. A method forthe production of such compounds can be achieved by optimising enzymesthat already is used as biocatalyst exemplified by the well-knownfamilies of lipases or other membrane independent enzymes. However, innature many of the enzymes catalysing the transfer of acylgroups areintegral membrane proteins. Among the membrane independentacyltransferases present in nature the vast majority catalyses anacyl-CoA dependent reaction. Both these classes of acyltransferases arenot suited as a biocatalyst in industrial methods since integralmembrane protein are not functioning in cell free systems and acyl-CoAis a to costly substrate. Furthermore, in applications involving enzymesbelonging to the lipase family the interesterification is dependent on awater free system. Hence, membrane independent acyltransferases thatcould use acyl-lipids as acyl donors in industrial methods for themanufacturing of fatty acid esters are limited today and no such enzymeis available which can manufacture several different fatty acid esterand/or fatty acid thioesters, i.e., use a lot of different acyl donorsand acyl acceptors.

There are also needs for enzymes to be used to improve the properties ofcomplex raw material. For example within the area of food production,modification of different components such as lipids present in food rawmaterial such as milk cereals, vegetables, eggs, vegetable oils, meat,fish, etc is desirable. Examples of improvements achieved by suchmodifications are enhanced emulsifying properties, increased shelf life,less off-flavour, etc. For example in many food applications enhancedemulsifying properties are desirable and can be achieved by convertingphospholipids (i.e. lecithin) present in the food raw material intolysophospholipids. Lipases are commonly used in such applicationsresulting in elevated levels of lysophospholipids but also unesterifiedfatty acids that can result in off-flavours. Conversion of phospholipidsinto lysolipids without increased amounts of unesterified fatty acids istherefore desirable and can be achieved with acyltransferases thattransfer the fatty acid from the phospholipid to an acyl acceptor suchas monoacylglycerols, diacylglycerols, alcohols, or any other acylacceptors present in or added to the raw material.

BRIEF DISCLOSURE OF THE INVENTION

Accordingly, in a first aspect the invention relates to one or morenucleotide sequence(s), derived from a nucleotide sequence encoding anacyltransferase polypeptide comprising at least one membrane-spanningregion, encoding an improved active membrane independent acyltransferasepolypeptide in which at least one amino acid residue of themembrane-spanning region has been deleted and/or substituted as comparedto the original acyltransferase polypeptide, wherein the encoded activemembrane independent acyltransferase polypeptide can produce lysolipidsand fatty acid esters and/or fatty acid thioesters such aslysophosphospholipid, lysolecithin, triacylglycerols, diacylglycerols,monoacylglycerols, phospholipids, glycolipids, waxesters, acylatedcarbohydrates and acylated amino acids. Such an improved acyltransferasecan be used in a huge number of chemical reactions for the production ofa large number of different fatty acid esters and/or fatty acidthioesters, which enables the possibility to in a economic way produce alarge amount of a single enzyme which then can be used for severalpurposes.

Additionally, such an acyltransferase, which is capable of catalysingseveral reactions, enables the possibility to facilitate the productionof a number of fatty acid esters and/or fatty acid thioesters by onesingle acyltransferase. Such an active membrane independentacyltransferase polypeptide may be used in a bioreactor for theproduction of desired fatty acid esters or as additive in food rawmaterial for modification of its lipid composition without the need of amicroorganism or a lipid membrane for the maintenance of theacyltransferase activity.

In another aspect, the invention relates to a nucleotide sequencemolecule comprising at least one promoter region which functions in ahost, the promoter region is operably linked to at least one nucleotidesequence as described above, which is operably linked to at least onenon-translated region which functions in a host.

In a further aspect, the invention relates to a method for theproduction of an active membrane independent acyltransferase polypeptidecomprising the steps of providing a host cell and a growth mediumpreparing a host cell culture, culturing the host cell culture andharvesting the host cell culture and recovering the polypeptide.

By providing a nucleotide sequence encoding a membrane independentacyltransferase without the ability to become integrated into a membraneand having the ability to utilise different acyl donors and acylacceptors, the ability to manufacture acylated products by a sole enzyme(i.e. fatty acid esters) is increased.

The membrane independent acyltransferase may be used in applicationssuch as cosmetics, pharmaceuticals, foods, food additives, candles,soaps, detergents, laundries, polymers, coatings, plasticizer, dryingoils, lubricants, varnishes, linoleum, printing, inks, textile dyes andsurfactants, especially within the area of synthesis of stereo specificisomers, which not is possible with the use of conventional organicsynthesis.

Furthermore, the synthesis of fatty acid esters with the use of such anenzyme in a cell free method, such as in a bioreactor can be moreefficient and less restricted since the method is only limited to theconditions by which the enzyme is active, whereas in a fermentationmethod the limitations is set by the conditions for the maintenance ofthe microorganisms. In such a fermentation system the fatty acid esterproducts to be synthesised is limited to the building components, suchas acyl donors and acyl acceptors present within the cell, whereas in acell free system the limitation is only set by the properties of theenzyme such as substrate specificity. In a cell free system it is easyto calculate the amounts of the building components which are necessaryto add to obtain an optimised enzyme catalysed method in which most ofthe building components ends up in the desired products such as fattyacid esters. Moreover, the use of lipases in a method for the synthesisof fatty acid esters is limited to water free conditions whereasmembrane independent acyltransferases catalyses the acyl transfers inwater containing systems.

Furthermore, use of a membrane independent acyltransferase as comparedto a microorganism for the synthesis of for example lysophospholipidsand/or fatty acid esters reduces the need of removing the microorganismafter the synthesis is finalised

By the use of the new improved enzyme according to the invention it ispossible to produce structured lipids without the need of organicsolvents, which would be both environmentally favourable, healthier andeliminates one or more purification steps after the production of thestructured lipids. Additionally, it may be easier to get an approval bythe authorities for such a product, manufactured in a process withoutthe use of organic solvents.

DESCRIPTION OF THE DRAWINGS

The invention is illustrated with reference to the drawings in which

FIG. 1 shows Western blot analysis using protein extract of the cellfree supernatant from growth of Pichia pastoris KM71H transformed withthe pATWAX construct.

FIG. 2 shows the synthesis of triacylglycerol catalysed by the membraneindependent acyltransferase (ATWAX), as visualized by autoradiography oflipid products separated on TLC.

FIG. 3. shows the time course of the wax esters synthesises from addedsoy lecithin and 13c-docosenoyl-alcohol (◯) or ricinoleoyl-alcohol (●)in cell free medium of Pichia pastoris cultures expressing the membraneindependent acyltransferase HisATWAX as described in EXAMPLE 5.

FIG. 4. shows the dependence of the ratio of the lecithin andricinoleoyl-alcohol substrates (panel A) and increased substrateconcentration with a fixed ratio of the substrates (panel B) on the waxester synthesis were determined as described in EXAMPLE 5.

FIG. 5. shows acyl group composition of soy lecithin (filled bars) andwax esters (open bars) produced from soy lecithin and ricinoleoylalcohol as described in EXAMPLE 5. Abbreviations used; palmitoyl (16:0),palmitoleoyl (16:1), stearoyl (18:0), oleoyl (18:1), linoleoyl (18:2),linolenoyl (18:3).

FIG. 6. shows microsomes prepared from wild type Saccharomycescerevisiae cells, overexpressing the yeast PDAT gene LRO1, catalysessynthesis of triacylglycerols but not wax esters. Acyltransferaseactivities were analysed in the presence of the substratesn1-oleoyl-sn2-[¹⁴C]oleoyl-phosphatidylcholine (lane 1) together witheither ricinoleoyl alcohol (Ric-OH, lane 2) or 13c-docosenol (22:1-OH,lane 3) as described in EXAMPLE 6.

FIG. 7 shows the alignment of Saccharomyces cerevisiae phospholipid:diacyiglycerol acyltransferase (ScPDAT) amino acid sequence (SEQ IDNO:3), encoded from the LRO1 gene, with the Schizosaccharomyces pombeSpPDAT (SEQ ID NO: 17), Arabidopsis At67O4 (SLO TD NO:5), At1254 (SEQ IDNO:13), At3O27 (SEQ ID NO:9), At4557 (SEQ ID NO:11) and the Crepisalpina Cp67O4 (SEQ ID NO:19) and Cp1254 (SEQ ID NO:20) deduced aminoacid sequences.

FIG. 8 shows part of the Saccharomyces cerevisiae phospholipid:diacyiglycerol acyltransferase (ScPDAT) amino acid sequence (SEQ IDNO:30), encoded from the LRO1 gene, aligned with amino acid sequencestranslated from the AnPDAT (SEQ ID NO:31) and AfPDAT (SEQ ID NO:32)nucleic acid sequences.

FIG. 9. shows the synthesis of triacylglycerol catalysed by the membraneindependent acyltransferase (HisATWAX-P6), as visualized byautoradiography of lipid products separated on TLC.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the context of the present application and invention the followingdefinitions apply:

The term “nucleotide sequence” is intended to mean a sequence of two ormore nucleotides. The nucleotides may be of genomic, cDNA, RNA, semisynthetic or synthetic origin or a mixture thereof. The term includessingle and double stranded forms of DNA or RNA.

The term “deleted and/or substituted” is intended to mean that one ormore amino acid residue(s) is/are removed (deleted) from the polypeptideand/or changed (substituted) into another amino acid(s).

The term “nucleotide sequence molecule” is intended to indicate aconsecutive stretch of three or more regions of nucleotide sequences.The nucleotide sequence molecule comprises a promoter region, anucleotide sequence and a non-translated region. The nucleotide sequenceor nucleotide sequence molecule may be of genomic, cDNA, RNA,semi-synthetic or synthetic origin, or a combination thereof. Thenucleotide sequence molecule is designed to express a nucleotidesequence located within the nucleotide sequence molecule when thenucleotide sequence molecule integrated into the genome or within amicroorganism.

The term “promoter region” is intended to mean one or more nucleotidesequences involved in the expression of a nucleotide sequence, e.g.promoter nucleotide sequences, as well as nucleotide sequences involvedin regulation and/or enhancement of the expression of the structuralgene. A promoter region comprises a promoter nucleotide sequenceinvolved in the expression of a nucleotide sequence, and normally otherfunctions such as enhancer elements and/or signal peptides. The promoterregion may be selected from a plant, virus and bacteria or it may be ofsemi-synthetic or synthetic origin or a mixture thereof as long as itfunctions in a microorganism. Example of a promoter region is themethanol oxidase promoter, which can be used for the expression ofpolypeptides in Pichia pastoris.

The term “a non-translated region” also called termination region isintended to mean a region of nucleotide sequences, which typically causethe termination of transcription and the polyadenylation of the 3′region of the RNA sequence. The non-translated region may be of nativeor synthetic origin as long as it functions in a microorganism accordingto the definition above.

The term “operably linked” is intended to mean the covalent joining oftwo or more nucleotide sequences by means of enzymatic ligation, in aconfiguration which enables the normal functions of the sequencesligated to each other. For example a promoter region is operably linkedto a signal peptide region and/or a coding nucleotide sequence encodinga polypeptide to direct and/or enable transcription of the codingnucleotide sequence. Another example is a coding nucleotide sequenceoperably linked to a 3′ non-translated region for termination oftranscription of the nucleotide sequence. Generally, “operably linked”means that the nucleotide sequences being linked are continuously and inreading frame. Linking is normally accomplished by ligation atconvenient restriction sites. If such sites do not exist, syntheticadaptors or the like are used in conjunction with standard recombinantDNA techniques well known for a person skilled in the art.

The term “acyltransferase” is intended to mean a polypeptide, which havethe ability to catalyse the transfer of an acyl group from one moleculeto another (i.e. interesterification). This transfer involves thebreakage of an ester or a thioester bound of the donor molecule and theformation of an ester or thioester bound between the transferred acylgroup and the acceptor molecule. Hence, in principal any molecule withan ester/thioester-linked acylgroup can act as a donor molecule and amolecule with at least one hydroxy or a thiol group could act as anacceptor molecule. Commonly occurring donor molecules are acyl-CoA orlipids such as phospholipids and the acyltransferases are in natureknown to catalyse e.g., with diacylglycerols, sterols and alcohols asacceptor molecules, the final step in the synthesis of the storagecompounds triacylglycerols (TAG), steryl esters and wax esters,respectively.

The term “lipid dependent acyltransferase” is intended to mean anacyltransferase as described above restricted to utilising lipids suchas phospholipids, glycolipids, triacylglycerols or other acyl-lipidsthat could serve as the acyl donor in the acyltransfer reaction. Thelecithin: cholesterol acyltransferase (LCAT) (Jonas A., 2000, Biochem.Biophys. Acta 1529: 245-256) and the bacterial glycerophospholipid:cholesterol acyltransferase (GCAT) (Brumlik and Buckley, 1996, J.Bacteriol. 178: 2060-2064) are the only known lipid dependent acyltransferase that has been shown to be functionally active as solubleproteins. All other known lipid dependent acyltransferases arepolypeptides with one or several membrane spanning regions and isexemplified by the phospholipid: diacylglycerol acyltransferase (PDAT)and its homologues. It should also be noted that the LCAT enzyme isdependent on an apolipoprotein for functionality. The bacterial GCATdoes not show any strong sequence homologies to neither the LCAT nor thePDAT enzymes or to any other known acyltransferases.

The term “membrane independent acyltransferase” are intended to mean anacyltransferase, which is functionally active without being via amembrane-spanning region integrated into a membrane. The “membraneindependent acyltransferase” is also active in a water-basedenvironment.

The term “enzymatic conditions” are intended to mean that any necessaryconditions available in an environment, which will permit the enzyme tofunction.

The term “membrane spanning region” is intended to mean part of apolypeptide which anchor the polypeptide into a membrane and ishydrophobic, i.e., the membrane spanning region, such as amino acidresidue number 80-96 of the polypeptide shown in SEQ ID NO:1 in thepatent application WO 00/60095, as predicted by a hydrophobic plot(Kyte, & Dolittle).

The term “stringent conditions” is intended to mean hybridisation andwashing conditions which permits the hybridisation between relatednucleotide sequences to be permitted during the hybridisation and remainhybridised during the washing, such as an overnight hybridisation at 42°C. in a solution comprising 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulphate and 20 mg/ml denatured sheared salmonsperm DNA followed by washing the hybridisation membrane or support in0.1×SSC at approximately 65° C.

The term “acyl donors” are intended to mean phospholipids,triacylglycerols or other molecules containing at least one esterifiedacyl group that can be donated to an acyl acceptor in the production offatty acid esters and/or fatty acid thioesters.

The term “acyl acceptors” are intended to mean molecules with at leastone hydroxy or thiol group, to which acyl groups derived from the acyldonors can be esterified in the formation of an fatty acid esters orthioesters.

The term “fatty acid esters” are intended to mean fatty acid estersproduced by a membrane independent acyltransferase catalysing theformation of ester bounds as described herein from the above mentionedacyl donor and acyl acceptors. Examples of fatty acid esters areacyl-lipids such as triacylglycerols, diacylglycerols,monoacylglycerols, phospholipids, glycolipids, lysolipids etc; waxesters(i.e. fatty acids esterified with long chain alcohols); acylatedcarbohydrates; acylated amino acids; or any other molecules with atleast one acyl group esterified to a hydroxyl group.

The term “stabiliser” is intended to mean any kind of stabilising agentused by persons skilled in the art in order to increase the stabilityand shelf life time of enzymes.

Nucleotide Sequences Nucleotide Sequence Molecules or Vectors of theInvention

The invention relates to one or more nucleotide sequence(s), derivedfrom a nucleotide sequence encoding an acyltransferase polypeptidecomprising at least one membrane-spanning region, encoding an improvedactive membrane independent acyltransferase polypeptide in which atleast one amino acid residue of the membrane-spanning region has beendeleted and/or substituted as compared to the original acyltransferasepolypeptide, wherein the encoded active membrane independentacyltransferase polypeptide can produce lysolipids and fatty acid estersand/or fatty acid thioesters such as lysophosphospholipid, lysolecithin,triacylglycerols, diacylglycerols, monoacylglycerols, phospholipids,glycolipids, waxesters, acylated carbohydrates and acylated amino acids.By deletion and/or substitution of one or more amino acid residues theencoded polypeptide looses the ability to become integrated into amembrane and remains membrane independent as compared to the originalpolypeptide. The numbers and/or the location of the amino acidresidue(s) to be deleted and/or substituted is/are not critical as longas the polypeptide by the deletion and/or substitution become membraneindependent. Part of the membrane-spanning region may be present as longas it does not integrate or attach the polypeptide to a membrane. Thepolypeptide encoding the membrane independent acyltransferase namedATWAX is a membrane independent acyltransferase which may be encoded bya nucleotide sequence, originally encoding an integral membrane proteinwith one or several membrane spanning regions wherein one or several ofthe membrane spanning regions has/have been deleted and/or substituted.The nucleotide sequence may also be synthetic or semi synthetic as longas it has the function of a membrane independent acyltransferase whichmay be used in the formation of fatty acid esters, like acyl-lipids suchas triacylglycerols, diacylglycerols, monoacylglycerols, phospholipids,glycolipids, lysolipids etc; waxesters (i.e. fatty acids esterified withlong chain alcohols); acylated carbohydrates; acylated amino acids; orany other molecules with at least one acyl group esterified to ahydroxyl group. The nucleotide sequence encoding the acyltransferase maybe derived from a nucleotide sequence encoding a lipid dependentacyltransferase polypeptide, such as a nucleotide sequence encoding anlipid dependent acyltransferase polypeptide catalysing an acyl transferreaction in which acylphospholipids acts as acyl donors, for example anucleotide sequence encoding a phospholipid: diacylglycerolacyltransferase.

Such nucleotide sequences may be obtained from different kind of speciessuch as bacteria, yeasts, fungi, plants, insects or mammalians. Examplesare Arabidopsis thaliana, Crepis palaestina, Euphorbia lagascae,Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillusstrains, e.g. A. niger, A. nidulans, A. fumigatus, A. sojae, Pichiastrains, such as P. Pastoris or P. methanolica Mucor strains, e.g. M.circinelloides, Hansenula, such as H. Polymorpha and Trichoderma,Klyveromyces, or Yarrowia. Examples on nucleotide sequences are shown inSEQ ID NO:1, 4, 8, 10, 12, 14, 15, 16, 18 or 20.

According to one embodiment the invention relates to a nucleotidesequence, wherein from 1 to 291 nucleotide sequence residue(s) has/havebeen deleted and/or substituted from the nucleotide sequence shown inSEQ ID NO:1. The number(s) of nucleotide sequence residues to be deletedis/are chosen in such a way that the open reading frame of thenucleotide sequence encoding the membrane independent acyltransferasepolypeptide is not disturbed and the membrane spanning regioncorresponding to nucleotide sequence 238 to 288 is deleted and/orsubstituted. One example is the nucleotide sequence shown in SEQ IDNO:2, where 290 nucleotide sequence residues have been deleted and/orsubstituted resulting in the membrane independent acyltransferasepolypeptide shown in SEQ ID NO:3.

According to another embodiment the invention relates to a nucleotidesequence, wherein from 1 to 219 nucleotide sequence residue(s) of the5′-end has/have been deleted and/or substituted from the nucleotidesequence shown in SEQ ID NO: 4, 1-87 nucleotide sequence residue(s) ofSEQ ID NO:8 and SEQ ID NO:10 and 1-190 nucleotide sequence residue(s) ofSEQ ID NO:12.

According to another embodiment the invention relates to a nucleotidesequence, wherein at least the nucleotide sequence residues 142 to 210have been deleted and/or substituted from the nucleotide sequence shownin SEQ ID NO:4, 19-87 nucleotide sequence residues of SEQ ID NO 8 andSEQ ID NO:10 and 130-190 nucleotide sequence residues of SEQ ID NO:12.

According to another embodiment the invention relates to a nucleotidesequence, wherein from 1 to 228 nucleotide sequence residue(s) of the5′-end has/have been deleted and/or substituted from the nucleotidesequence shown in SEQ ID NO: 16, 1-219 nucleotide sequence residue(s) ofSEQ ID NO:18 and 1-261 nucleotide sequence residue(s) of SEQ ID NO:20.

According to another embodiment the invention relates to a nucleotidesequence, wherein at least the nucleotide sequence residues 169 to 228have been deleted and/or substituted from the nucleotide sequence shownin SEQ ID NO:16, 151-219 nucleotide sequence residue(s) of SEQ ID NO:18and 193-261 nucleotide sequence residue(s) of SEQ ID NO:20.

The number(s) of nucleotide sequence residues to be deleted is/arechosen in such a way that the open reading frame of the nucleotidesequence encoding the membrane independent acyltransferase polypeptideis not disturbed and the membrane spanning region are removed/deleted.

According to one embodiment of the invention the nucleotide sequenceencoding the membrane independent acyltransferase polypeptide mayhybridise under stringent conditions to a nucleotide sequence as shownin SEQ ID NO:1, 2, 4, 6, 8, 10, 12, 14, 15, 16, 18 or 20. Furthermorethe nucleotide sequence as shown in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12,14, 15, 16, 18 or 20 may be different as compared to another nucleotidesequence due to the degeneracy of the genetic code.

Additionally the nucleotide sequence encoding the membrane independentacyltransferase polypeptide may at least show 75%, 80%, 85%, 90% or 95%homology to the amino acid sequence(s) shown in SEQ ID NO:3, 7, 9, 11,13, 17, 19, 21 or a homologue thereof.

Furthermore, the nucleotide sequence shown in SEQ ID NO 2, encoding themembrane independent acyltransferase polypeptide shown in SEQ ID NO 3,may be modified by removing (deleting) nucleotides, encoding one orseveral amino acid residues in the N-terminal part corresponding to thefirst 71 amino acid residues of the polypeptide shown in SEQ ID NO 3,with maintained acyltransferase activity. Furthermore one or more aminoacid residues may be substituted as long as the acyltransferase activityremains. Methods, which are suitable for the removal (deletion) of aspecific nucleic acid sequence are well known for a person skilled inthe art and includes methods such as PCR.

Moreover, the amino acid residues S229, D472, and H523 shown in SEQ IDNO 3 are essential for activity as described in the examples and is heresuggested to be part of the a catalytic triad in the active site of theenzyme.

Additionally, the invention relates to an oligonucleotide, whichspecifically hybridise under stringent conditions to the nucleotidesequence(s) and/or the nucleic acid molecule(s) described herein. Theoligonucleotide may be used for the detection of the nucleotide sequenceand/or the nucleotide sequence, such as the presence of the nucleotidesequence within a host cell.

According to another embodiment the invention relates to a nucleotidesequence molecule, which comprises at least one promoter region whichfunctions in a host. The promoter region is operably linked to at leastone nucleotide sequence as described above, which is operably linked toat least one non-translated region which functions in a host.Furthermore, a signal peptide may be present between the promoter regionand the nucleotide sequence as described above.

The nucleotide sequence molecule may be present in a vector, such as anexpression vector, which may be used for the production of thepolypeptide, which has acyltransferase activity. The vector is typicallyderived from plasmid or viral DNA. A number of suitable expressionvectors for expression in the host cells mentioned herein arecommercially available or described in the literature. Any kind ofvector may be used as long as it functions in a host cell which iscapable of performing glycosylation of the polypeptide, such as vectorswhich functions in yeast. Useful expression vectors for yeast cellsinclude the 2μ plasmid and derivatives thereof, the POTI vector (U.S.Pat. No. 4,931,373), the pJSO37 vector described in Okkels, Ann. NewYork Acad. Sci. 782, 202-207, 1996, and pPICZ A, B or C (Invitrogen).

Other vectors for use in this invention include those that allow thenucleotide sequence encoding the polypeptide to be amplified in copynumber. Such amplifiable vectors are well known in the art. Theyinclude, for example, vectors able to be amplified by DHFR amplification(see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp,“Construction Of A Modular Dihydrafolate Reductase cDNA Gene: AnalysisOf Signals Utilized For Efficient Expression”, Mol. Cell. Biol., 2, pp.1304-19 (1982)) and glutamine synthetase (“GS”) amplification (see,e.g., U.S. Pat. No. 5,122,464 and EP 338,841).

The vector may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question. When the host cell is a yeastcell, suitable sequences enabling the vector to replicate are the yeastplasmid 2μ replication genes REP 1-3 and start of replication.

The vector may also comprise a selectable marker, e.g., a gene theproduct of which complements a toxin related deficiency in the hostcell, such as the gene coding for dihydrofolate reductase (DHFR) or theSchizosaccharomyces pombe TPI gene (described by P. R. Russell, Gene 40,1985, pp. 125-130), or one which confers resistance to a drug, e.g.,ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin,hygromycin, zeocin or methotrexate. For Saccharomyces cerevisiae,selectable markers include ura3 and leu2. For filamentous fungi,selectable markers include amdS, pyrG, arcB, niaD and sC.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression ofthe polypeptide of the invention. Each control sequence may be native orforeign to the nucleic acid sequence encoding the polypeptide. Suchcontrol sequences include, but are not limited to, a leader sequence,signal peptide, polyadenylation sequence, propeptide sequence, promoter(inducible or constitutive), enhancer or upstream activating sequence,signal peptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter. Examples of suitable controlsequences for use in yeast host cells include the promoters of the yeastα-mating system, the yeast triose phosphate isomerase (TPI) promoter,promoters from yeast glycolytic genes or alcohol dehydrogenase genes,the ADH2-4c promoter, and the inducible GAL promoter. Examples ofsuitable control sequences for use in filamentous fungal host cellsinclude the ADH3 promoter and terminator, a promoter derived from thegenes encoding Aspergillus oryzae TAKA amylase triose phosphateisomerase or alkaline protease, an A. niger α-amylase, A. niger or A.nidulans glucoamylase, A. nidulans acetamidase, Rhizomucor mieheiaspartic proteinase or lipase, the TPI1 terminator and the ADH3terminator.

The presence or absence of a signal peptide will, e.g., depend on theexpression host cell used for the production of the polypeptide to beexpressed (whether it is an intracellular or extra cellular polypeptide)and whether it is desirable to obtain secretion. For use in filamentousfungi, the signal peptide may conveniently be derived from a geneencoding an Aspergillus sp. amylase or glucoamylase, a gene encoding aRhizomucor miehei lipase or protease or a Humicola lanuginosa lipase.The signal peptide is preferably derived from a gene encoding A. oryzaeTAKA amylase, A. niger neutral α-amylase, A. niger acid-stable amylase,or A. niger glucoamylase. For use in yeast cells suitable signalpeptides have been found to be the α-factor signal peptide from S.cereviciae (cf. U.S. Pat. No. 4,870,008), a modified carboxypeptidasesignal peptide (cf. L. A. Valls et al., Cell 48, 1987, pp. 887-897), theyeast BAR1 signal peptide (cf. WO 87/02670), the yeast aspartic protease3 (YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp.127-137), and the synthetic leader sequence TA57 (WO98/32867).

Furthermore the invention relates to an oligonucleotide, whichhybridises under stringent conditions (as defined above) to a nucleotidesequence and/or a nucleotide sequence molecule as described above.

Host Cells Method and Polypeptide of the Invention

Any suitable host cell may be used for the maintenance and production ofthe vector of the invention as long as the host is capable of producinga glycosylated product. The host cell may be a eukaryotic cell, forexample fungi, yeast, insects and mammalian cells. A eukaryotic systemmay provide significant advantages compared to the use of a prokaryoticsystem, for the production of certain polypeptides encoded by nucleotidesequence molecules and/or vectors present within the host cell orintegrated into the genome of the host cell. For example, yeast cangenerally be grown to higher cell densities than bacteria and may becapable of glycosylating expressed polypeptides, where suchglycosylation is important for a proper folding of the polypeptideand/or catalytic activity of the polypeptide.

The host cell may be a host cell belonging to a GMP (Good ManufacturingPractice) certified cell-line. Examples of suitable filamentous fungalhost cells include strains of Fusarium, Trichoderma, Aspergillus, e.g.A. oryzae, A. niger, A. sojae or A. nidulans, Mucor, e.g. Mcircinelloides. Examples of suitable yeast host cells include strains ofSaccharomyces, e.g. S. cerevisiae, Schizosaccharomyces, Klyveromyces,Pichia, such as P. Pastoris or P. methanolica, Hansenula, such as H.Polymorpha or Yarrowia. Examples of P. Pastoris strains are X-33, KM71H,and GS115 which may be obtained from Invitrogen Inc. Pichia pastoris isa methylotrophic yeast which can grow on methanol as a sole carbon andenergy source (Ellis et al., 1985). P. pastoris is also amenable toefficient high cell density fermentation technology. Therefore is Pichiapastoris a suitable host for expression of heterologous protein in largequantity, with a methanol oxidase promoter based expression system(Cregg et al., 1987). Additional suitable donor cell lines are known inthe art and available from public depositories such as the American TypeCulture Collection, Rockville, Md.

The vector is transferred (introduced) into the host cell using asuitable method dependent on which host cell has been selected. Theintroduction of the vector harbouring the nucleotide sequence moleculeinto fungal cells may be by a method involving protoplast formation,transformation of the protoplasts, and regeneration of the cell wall ina manner known per se. Suitable procedures for transformation ofAspergillus host cells are described in EP 238 023 and U.S. Pat. No.5,679,543. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology153: 163; Hinnen et al., 1978, Proceedings of the NationalAcademy of Sciences USA 75: 1920: and as disclosed by ClontechLaboratories, Inc, Palo Alto, Calif., USA (in the product protocol forthe Yeastmaker™ Yeast Transformation System Kit) or by using the PichiaManual supplied by Invitrogen Inc. These methods are well known in theart and e.g., described by Ausbel et al. (eds.), 1996, Current Protocolsin Molecular Biology, John Wiley & Sons, New York, USA.

In the production methods (process) of the present invention, the cellsare cultivated in a growth medium suitable for maintenance and/orproduction of the nucleotide sequence molecule and/or the vector usingmethods known in the art. For example, the cell may be cultivated byshake flask cultivation, small-scale or large-scale fermentation(including continuous, batch, fed-batch, or solid state fermentations)in laboratory or industrial fermenters performed in a suitable growthmedium and under conditions allowing the vector, nucleotide sequencemolecule or polypeptide to be expressed and/or isolated. The vector,nucleotide sequence molecule or the polypeptide may be used in thechemical or in the pharmaceutical industry. The cultivation takes placein a suitable growth medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable growthmedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). The cultivation of Pichia pastoris isperformed using the method described in EXAMPLE 2 or any other suitablemethod. After cultivation, the polypeptide is recovered from the culturemedium, the cells or after separating the cells from the culture medium.The recovered polypeptide encodes an active membrane independentacyltransferase without the ability to become integrated into amembrane, i.e., one ore more of the amino acid residue(s) present in themembrane spanning region has/have been deleted and/or substituted.Examples of methods are those mentioned in Maniatis et al., MolecularCloning: A Laboratory Manual (Cold Spring Harbour Press) (1989) andQiagen Inc.

According to another embodiment the polypeptide is an acyltransferaseactive at a pH ranging from about 4 to about 10, and stable at atemperature below about 60° C. The enzymatic activity of the polypeptidecan be measured using the assay method described in EXAMPLE 4 or EXAMPLE5.

The polypeptide may furthermore be immobilised to a carrier. Suitablecarriers and methods for the immobilisation of the polypeptide to thecarrier are well known for a person skilled in the art (Tisher, W., &Kasche, V., 1999, Trends Biotechnol. 17(8): 326-335).

According to one embodiment of the invention the polypeptide named ATWAXand described above may be lyophilised and/or freeze-dried.Lyophilisation and/or freeze-drying may be performed using conventionaltechniques known for a person skilled in the art.

A further embodiment relates to the use of a nucleotide sequence and/ora nucleotide sequence molecule and/or a vector and/or a host cell and/orthe methods and/or the polypeptide of the invention. The polypeptide maybe used for the production of fatty acid esters. Examples of fatty acidesters are acyllipids such as triacylglycerols, diacylglycerols,monoacylglycerols, phospholipids, glycolipids, lysolipids; waxesters;acylated carbohydrates; acylated amino acids; or any other moleculeswith at least one acyl group esterified to a hydroxyl group are fattyacid esters.

Examples of products which may be produced by the method are fatty acidesters used in cosmetics, foods, food additives, dairy products,confectionary, flavours, bakery, pharmaceuticals, candles, soaps,detergents, laundries, polymers, coatings, plasticizer-, drying oils,lubricants, varnishes, linoleum, printing, inks, textile dyes andsurfactants.

Examples of what the invented polypeptide may be used for are listedbelow.

Production of Structured Lipids

The invented polypeptide(s) are suitable to be used in the production ofstructured lipids without the need of organic solvents, which would beboth environmentally favourable, healthier and eliminates one or morepurification steps after the production of the structured lipids.Additionally, it may be easier to get an approval by the authorities forsuch a product, manufactured in a process without the use of organicsolvents. The positional distribution of acyl groups differing in lengthand degree of saturation in the triacylglycerol molecule is known to beimportant regarding nutritional and health aspects partly due todifferences in digestibility and absorbability. As an example mosttriacylglycerols of vegetable origin are highly unsaturated at the2-position, mainly oleic and linoleic acid. However, in human milk fatthe saturated fatty acid palmitic acid is highly enriched at the2-position and it is known that such type of fat is more easily absorbedand utilized by infants. Structured triacylglycerols mimicking theproperties of triacylglycerols in the human milk fat can be manufacturedin a process in which ATWAX catalyses the transfer of acyl groups fromthe sn2 position of lecithin to monoacylglycerol with palmitic acid atthe sn2 position (2-palmitoyl glycerol), yielding triacylglycerolsenriched with unsaturated fatty acids such as oleic and linoleic acid inthe 1-, 3-positions and palmitic acid in the 2-position. The source ofthe 2-palmitoyl glycerol for use in this process may be obtained by 1, 3specific lipase hydrolyses of palm oil enriched in triacylglycerols withpalmitic acid at the 2-position.

In another application structured lipids are used as fat replacers inlow calorie foods. Acylglycerols with an acetyl group at the 2-positionare used as fat replacers in low calorie foods such as dairy products,bakery, cereals, pasta, cheese, tofu, chocolate, chocolate confections,margarine, salted snacks, sour cream, spreads etc. This diet fat can bemanufactured in a process in which ATWAX catalyse the transfer of acylgroups from the sn2 position of lecithin to 2-acetyl glycerol. In asimilar manner, structured diacylglycerols can be produced by anacyltransfer of fatty acids from an acyldonor such as lecithin toglycerol as the acceptor molecule. The major product in such a processis 1,3-diacylglycerol (i.e. diacylglycerol with acyl groups at the sn1and sn2 position), since the ATWAX enzyme has preferences for theacylation of the sn 1 and sn3 positions of the glycerol molecule. psProduction of Fat-Soluble Molecules.

The invented polypeptide(s) are suitable to be used to render moleculesmore hydrophobic by coupling fatty acids via an acylation reaction tomolecules that otherwise are badly soluble in hydrophobic solvents suchas fats and oils. Example of such modification, is the acylation ofwater soluble flavours and vitamins which makes these fatty acid estersof flavours and vitamins more fat soluble and hence more suitable forcertain applications such as food, cosmetic, and pharma applications. Asan example fatty acid esters of vitamins such as vitamin E (tocopherol)are used in skin-care products since the vitamins are more readilyadsorbed into the skin. In certain food applications it is desirable tomake water-soluble flavours, vitamins or other additives are more easilymixed into fatty foodstuffs. In a process involving ATWAX themanufacturing of fatty acid esters by the acylation of hydrophilicmolecules possessing a hydroxyl group can be performed by anacyltransfer catalysed by ATWAX. The acyl donor molecule in thisreaction can be lecithin or phospholipids or any other suitableacyl-lipids.

Removal of Undesirable Fat.

The invented polypeptide(s) are suitable to be used enables the removalof one or more fatty acids from a molecule by the use of ATWAX, such asby transferring one or more fatty acids from a molecule to an acceptormolecule such as monoacylglycerol, diacylglycerol. Phospholipids presentwithin milk and dairy products are examples of molecules from which onefatty acid may be removed are. The major phospholipids present in milksfrom mammals are phosphatidylcholine, phosphatidylethanolamine andsphingolipids, each comprising about 30% of the total phospholipidspresent. These phospholipids are part of the milk fat globule membranefraction, which constitutes a minor part of the whole milk lipids. Apartfrom the phospholipids this membrane lipid fraction also contains TAG,diacylglycerol and monoacylglycerol.

It is desirable to remove fatty acids, especially unsaturated fattyacids such as oleic, linoleic, and linolenic acid from milk prior to useof the milk in products such as low fat or non fat products. Duringstorage the unsaturated fatty acids that are mainly present on thesn2-position of the phospholipids, becomes oxidised and thus the milkproduct becomes rancid with a bad smell and taste (off-flavour). Today,there is no suitable method for removal of these undesirable fatty acidsfrom milk. By the addition of ATWAX to milk the fatty acid on the sn2position of the phospholipid is transacylated to the acceptor moleculesuch as monoacylglycerol and/or diacylglycerol by which triacylglycerolis produced. This formed TAG will be removed together with the main TAG,in the production of low fat or non-fat product such as dry milk powder,cheese, yoghurt and other dairy products. Thereby the off-flavouring isreduced and/or eliminated and the shelf life time of the products couldbe increased.

Furthermore, in the transfer of fatty acids in milk from thephospholipids to acceptor molecules such as monoacylglycerols ordiacylglycerols, the phospholipids will be converted tolysophospholipids. With an increased fraction of lysophospholipids, themembrane lipid fraction is more easily disintegrated and theencapsulated TAG is released. This released fraction of TAG as well asthe TAG that is formed in the transfer of fatty acids from lecithin tomonoacylglycerols and/or diacylglycerol will be removed together withthe main TAG fraction. Thereby a process, in production of low-fat ornon-fat milk products, involving the use of ATWAX can more efficientlyreduce the fat content in such milk products.

Another field of application is to use ATWAX to remove phospholipidse.g. lecithin. In the refining of vegetable oils, the removal of thelecithin fraction, i.e. degumming is an important process in theproduction of high quality oils. In a refining process involving the useof ATWAX, lecithin present in the oil can be converted intolysolecithin, which will be removed from the oil into the water phase.The fatty acid removed from the lecithin in this process will betransferred to an acceptor molecule present in the oil such asdiacylglycerol by which triacylglycerol is formed.

Modification of Lipids Presents in Animal and Plant Raw Material.

In the field of baking, bread improvers such as emulsifiers based onlipids are commonly used. However, these emulsifiers are known to giveoff-flavour and also caking and lumping problems, especially in hot andhumid climates. In flour, such as wheat flour, polar lipids mainlylecithin and galactolipid (e.g. digalctosyldiacylglycerol) are present.In a baking process in which ATWAX is added, the lecithin and thegalactolipid deriving from the flour, can enzymatically be convertedinto the corresponding lysolipids. This conversion of the polar lipidsinto lysolipids are known to give a similar stabilising effect of thedough as what is achieved with the today commonly added emulsifiers suchas diacetyl tartaric acid esters of monoacylglycerols. Therefore theATWAX enzyme can totally or partly replace the use of emulsifiers in thebaking process and thus reduce the problems with off-flavour and thetendency of lump formation.

In a similar manner the conversion of lecithin or phospholipids presentin “raw materials” such as milk, flour, eggs, soy protein, cocoa, or anyother animal or plant materials into lysolecithin or lysophospholipidscan be executed in a process involving ATWAX. In such as processimportant properties of the raw material are modulated, such asamphiphilic nature, texture, melting point, viscosity, flavour,emulsification, foaming, and wetting, to be suited for the production ofa certain complex foodstuffs. Thereby, the need for food additives suchas emulsifiers, wetting agents, dough strengtheners, and film formersare reduced.

Kit of the Invention

A kit comprising the polypeptide which has the enzymatic activity of a(membrane independent) acyltransferase and the membrane spanning regionremoved or a fragment thereof or a kit in which the polypeptide has beenimmobilised on a carrier. The polypeptide may be provided in the kit aslyophilised or freeze dried. The kit may also comprise components, whichare essential for the stability and activity of the polypeptide, such asa stabiliser. The kit may furthermore comprise a manual withinstructions for the use of the polypeptide.

The following examples are intended to illustrate but not to limit theinvention in any manner, shape, or form, either explicitly orimplicitly.

EXAMPLES Example 1

Amplification of Nucleotide Sequences, Homologues to the Saccharomveescerevisiae Gene LRO1, for Expression in Pichia pastoris.

Nucleotide sequences were amplified from a plasmid template(pBluescript, Stratagene Inc.) containing the intact yeast gene LRO1,encoding a phospholipid: diacylglycerol acyltransferase (PDAT) with onemembrane-spanning region, (described in Dahlqvist et al., 2000, PNAS97:6487-6492) by thermo stable Pfu Turbo Polymerase (Stratagene Inc.). Aset of primers was designed for the amplification of three differentnucleotide sequences A, B, and C, identical in nucleotide sequence tothe part of the LRO1 gene encoding the amino acid residues 98 to 661,170 to 661 and 190 to 661 respectively. The forward (5′ end) primersused for this PCR reaction were for the sequences A, B, and C;5′CCATGGGAATGAATTCATGGCTTATCATGTTCATAATAGCGATAGC3′ (SEQ ID NO; 22),5′CCATGGGAATGAATTCCGAGGCCAAACATCCTGTTGTAATG3′ (SEQ ID NO: 23), and5′CCATGGGAATGAATTCGGAGTTATTGGAGACGATGAGTGCGATAGT3′(SEQ ID NO:24)respectively.

The oligonucleotide sequence, 5′GCCTCCTTGGGCGGCCGCTCACATTGGGAGGGCATCTGAGAAAC3′ (SEQ ID NO: 25) was usedas the reverse (3′ end) primer in all the three PCR amplifications. Thethree amplified nucleotide sequences all lack the sequence region,present in the LRO1, which encodes a transmembrane region. The amplifiednucleotide sequence A is shown in SEQ ID NO 2.

Additionally one nucleotide sequence was amplified resulting in thenucleotide sequence D with nucleotides encoding 6-residue histidine atthe N-terminus in frame with the region encoding the amino acid residues98 to 661 of the yeast PDAT. This was achieved by first sub cloning theamplified nucleotide sequence A above into the NheI and XhoI sites ofthe plasmid pET28a(+) and then using the following oligonucleotideprimers for PCR amplification; 5′CCATGGGAATGAATTCATGGGCAGCAGCAGCCATCATCAT3′ (SEQ ID NO: 26) and 5′GCCTCCTTGGGCGGCCGCTCACATTGGGAGGGCATCTGAGAAAC3′(SEQ ID NO: 27).

The amplified PCR products A, B, C, and D above, were purified, digestedby EcoRI and NotI, and subcloned between the EcoRI and NotI sites of thePichia expression vector PpicZ□A in frame with the sequence encoding the□□factor signalpeptide present in the expression vector. The resultantPichia expression vectors are named pATWAX, p72ATWAX, p92ATWAX, andpHISATWAX, with inserts encoding the polypeptides ATWAX, 72ATWAX,92ATWAX, and HISATWAX respectively. These vectors were linearized usingunique SacI restriction site for transformation in Pichia pastoris hoststrain.

Site directed mutagenesis of the ATWAX polypeptide sequence described inSEQ ID NO: 2 were performed in order to identify the catalytic triad.The PCR based mutagenesis were performed using mega-primer method (Ling,M. M., & Robinson, B. H., 1997, 254(2): 157-178) for the construction ofthree nucleotide sequences encoding ATWAX-S229A, ATWAX-D472N andATWAX-H523A with the single residue mutant S229A, D472N and H523A,respectively.

Example 2

Transformation in Pichia and Growth for Expression.

Competent Pichia pastoris cells were prepared according to the procedurementioned in the EasySelect Manual supplied by Invitrogen Inc.Electroporation, as described in the EasySelect Manual, was used totransform the linearized expression vector, pATWAX, p72ATWAX, p92ATWAXor pHISATWAX described in EXAMPLE 1 above, into the Pichiapastoris hoststrain X-33 or KM71H. The procedure of Zeocin selection was used toselect transformants, which were plated on YPD medium containing Zeocin.For the expression of the transformed genes, cells were initiallycultured to a final O.D. of 3-5 in BMGY medium supplemented with 1%(v/v) glycerol, after which cells were subsequently washed with eithersterile water or YPD medium. The washed cells were then suspended inBMMY medium supplemented with 0.5% (v/v) methanol for induction of thetransgene and further cultured for 3-4 days in a volume corresponding to0.5-0.2 of the original volume. Methanol (20%, v/v) was added to a finalconcentration of 0.5% (v/v) every 24 hours. Cell-free medium wascollected by centrifugation and was used for western blot analyses andenzyme activity studies.

Example 3

Western Blot Analysis of Cell Free Medium of Pichia pastoris KM71HTransformed with pATWAX.

In order to determine the presence of ATWAX in the cell free culturemedium, P. pastoris KM71H transformed with pATWAX were cultured asdescribed in EXAMPLE 2. Aliquots of cell free culture medium werewithdrawn at different time points following induction and subjected toWestern blot analysis using anti yeast-PDAT polyclonal antibody. Theantibody was raised in a rabbit by the injection of partially purifiedATWAX. The ATWAX used for this purpose was produced in Echerichia coli.

The western blot based on immunodetection system as presented in FIG. 1clearly show the presence of a polypeptide, present in the cell freemedium with a molecular weight of approximately 82 kDa, thatcross-reacts with the anti-yeast PDAT. By comparing the results obtainedon the western blot analyses obtained with the cell free medium from 58,82 and 112 hours of induction (i.e. FIG. 1 lane 1, 2, and 3,respectively) it is concluded that the secreted ATWAX is continuouslysecreted and accumulated in the cell free medium up to at least 112hours of induction without being degraded. This is further supported bythe lack of additional band of lower molecular weight that could bereferred to as degradation products. Cell free medium of theuntransformed Pichia strain did not crossreact with the ATWAX antibody(laneS). These data also indicates that the ATWAX present in the cellfree medium is glycosylated and that the glycosylation contributes toabout 17 kDa of the molecular weight, since a non-glycosylated ATWAXshould have a weight of 65 kDa as calculated from its amino acidcomposition.

Example 4

Detection of ATWAX Enzyme Activity in the Culture Supernatants.

To determine the enzyme activity in the cell free culture medium thecell free supernatant was assayed for enzyme activity as follows. Thecell free supernatant of the induced cultures described in EXAMPLE 2,from 116 hours of growth of Pichia pastoris KM71H transformed with thepATWAX construct encoding ATWAX, the polypeptide described in SEQ ID NO:2 (FIG. 2, lane 2) and the congenic wt strain (FIG. 2, lane 1) wereassayed for acyl transferase activity. Lipid substrate, sn1-palmitoylsn2-[¹⁴C]linoleoyl-phosphatidylethanolamine (5 nmol; 5000 dpm/nmol) anddioleoylglycerol (2.5 nmol) dissolved in chloroform was aliquoted in 1.5ml tubes and the chloroform was evaporated under a stream of N₂ (g).After addition of 20 ul 0.25 M potassium phosphate, pH 7.2, the mixturewas violently agitated and 80 ul of cell free supernatant was added andincubated at 30° C. for 90 min. Lipids were extracted from the reactionmixture into chloroform (Bligh, E. G. and Dyer, W. J (1959) Can. J.Biochem. Physiol. 37, 911-917) and separated by TLC on silica gel 60plates (200×200 mm) in chloroform/methanol/acetic acid/water(85:15:10:3.5) migrating 90 mm using an automatic developing chamber(Camag). The plate was dried and redeveloped in hexane/diethylether/acetic acid (80:20:1) with a solvent migration of 180 mm. Theradioactive lipids were visualized and quantified on the plates byelectronic autoradiography (Instant Imager; Packard). As a control, theenzyme activity in the cell free supernatant of the wild type hoststrain culture was analysed. As shown in FIG. 2, the majority of the[¹⁴C]linoleoyl group translocated from phosphatidylethanolamine isassociated with triacylglycerol after the incubation. This demonstratesthat the truncated membrane independent form of yeast PDAT, referred toas a membrane independent acyltransferase that we have named ATWAX, isable of catalysing the formation of TAG by an acyltransfer fromphosphatidylethanolamine to diacylglycerol (DAG). Radiolabeledacylgroups can also be detected as unesterified fatty acids indicatingthe presence of a lipase activity. However, since a release ofradiolabeled fatty acids also occur in the cell free supernatant ofuntransformed host strain it is not possible to conclude whether thislipase activity is associated with ATWAX.

The cell free supernatant of the induced cultures of P. pastoris KM71Htransformed with the p72ATWAX or p92ATWAX constructs were by westernblot analyses shown to be expressed and secreted in to the culturemedium. However the cell free medium containing these truncatedpolypeptides, lacking a stretch of 72 or 92 amino acids residues of theATWAX N-terminus, respectively, did not catalyse the synthesis of TAGwhen analysed for enzyme activity according to method described above.

Furthermore, nucleotide sequences encoding the ATWAX-S229A, ATWAX-D472Nand ATWAX-H523A mutant polypeptides were generated as described inEXAMPLE 1 and expressed in Pichia pastoris as described in EXAMPLE 2.The expression of these mutant polypeptides was verified by western blotanalyses of aliquots of cell free medium from cultures expressing thesepolypeptides, respectively. However, all three mutant polypeptides wereinactive when assayed for acyltransferase activity according to methoddescribed above. Hence, the amino acid residues S229, D472 and H523 areessential for the catalytic activity and are therefore here suggested tobe part of a catalytic triad.

A membrane independent acyltransferase with an N-terminal stretch of sixHistidine residues was produced by the expression of the constructHisATWAX in Pichia pastoris (described in EXAMPLE 1 and 2). Thispolypeptide was when analysed for acyltransferase activity as describedabove shown to be active with similar catalytic properties as the ATWAX.

Example 5

Production of Waxesters

Wax esters can be synthesised from soy lecithin and different alcoholsby the catalyses of the membrane independent acyltransferases, ATWAX andHisATWAX. This acyltransferases was produced and secreted into theculture medium by expressing the construct pATWAX or pHisATWAX,respectively in Pichia pastoris (described in EXAMPLE 2). Aliquots ofcell free medium was prepared and stored at −20° C.

The ability to synthesise wax esters from soy lecithin and13c-docosenoyl-alcohol (◯ in FIG. 3) or ricinoleoyl-alcohol (● in FIG.3) by the membrane independent His-tagged acyltransferase present in thecell free culture medium was investigated and the results are given inFIG. 3. The conditions for the synthesis were as follows; lecithin (2.5mg), sn1-oleoyl-sn2-[¹⁴C]oleoyl-phosphatidylcholine (5 nmol; 5000dpm/nmol) and 6 mmol of 13c-docosenoyl-alcohol or ricinoleoyl-alcoholdissolved in chloroform was aliquoted in 12 ml glass tubes and thechloroform was evaporated under a stream of N₂ (g). To the dry lipidsubstrate 0.5 ml of cell free medium and 25 ul 1.0 M potassiumphosphate, pH 7.2 were added. The reaction mixture was sonicated in awater bath (Branson 2510) for 30 min and was further incubated at 37° C.to a final incubation time as indicated in FIG. 3. Lipids were extractedfrom the reaction mixture into chloroform (Bligh, E. G. and Dyer, W. J(1959) Can. J. Biochem. Physiol. 37, 911-917) and separated by TLC onsilica gel 60 plates (200×200 mm) in hexane/diethyl ether/acetic acid(55:45:0.5) with a final solvent migration of about 180 mm. Wax estersproducts were verified through the methylation of the wax ester productsexcised from the TLC plate (using method described in Dahlqvist et al.,2000, PNAS 97: 6487-6492) followed by the separation of the methylationproducts on silica gel 60 plates in hexane/diethyl ether/acetic acid(55:45:0.5). Only two components were detected, methyl esters of fattyacids and free alcohols as identified by means of appropriate standards.The amounts of wax esters produced, from the added radiolabeledsn1-palmitoyl sn2-[¹⁴C]linoleoyl-phosphatidylcholine and thenon-labelled alcohols, were quantified on the plates by electronicautoradiography (Instant Imager; Packard) as percentage of radiolabel inwax esters of total added. As shown in FIG. 3 the ATWAX was catalysingthe synthesis of wax esters of 13c-docosenoyl-alcohol (◯ in FIG. 3) orricinoleoyl-alcohol (● in FIG. 3) with similar efficiencies, whichreached a plateau after 4 hours of incubation at which about 55-60% ofthe radiolabeled acylgroups of the added phosphatidylcholine had formeda wax ester with the alcohol. Apart from the formation of the wax ester,lysophospholipids is also formed in this reaction.

The dependence of the ratio of the added lecithin and alcohol substrateon the conversion rate is presented in FIG. 4A. The conditions for thesynthesis were as follows; lecithin (10 mg),sn1-oleoyl-sn2-[¹⁴C]oleoyl-phosphatidylcholine (10 mmol; 5000 dpm/nmol)and 3.7, 7.4, 11.1, or 18,7 mg of ricinoleoyl-alcohol, giving an alcoholto lecithin ratio as indicated in FIG. 4A, dissolved in chloroform wasaliquoted in 12 ml glass tubes and the chloroform was evaporated under astream of N₂ (g). To the dry lipid substrate 1.2 ml of cell free mediumand 60 ul 1.0 M potassium phosphate, pH 7.2 was added. The reactionmixture was sonicated in a water bath (Branson 2510) for 30 min and wasfurther incubated at 37° C. to a final incubation of 4 hours. Lipidswere extracted and analysed as described above. These analyses show thatat a weight ratio of the alcohol to lecithin of 0.4 (corresponding to anequimolar amounts of alcohol and lecithin added) 28% of the radiolabeledfatty acids was converted into waxesters and by increasing the ratio5-fold the wax ester synthesis was increased 2-fold.

By increased substrate concentration with constant lecithin to alcoholweight ratio, the conversion into waxesters is decreased as shown inFIG. 4B. Incubating lipid substrates as described above together with1.2 ml of cell free medium at 37° C. for 20 hours performed theseanalyses. From the results presented in FIG. 4 A and B it is evidentthat in order to optimise the yield of waxesters produced from lecithinand an alcohol the total substrate concentration and the substrate ratioare important factors to consider.

The major lipid component in the soy lecithin is phosphatidylcholine andphosphatidylethanolamine constituting about 60% of the acyl lipidspresent, other phospholipids present are phosphatidylinositol,phosphatidylglycerol and phosphatidic acid, which contributes up toapproximately 25%, the remaining lipids are neutral lipids, lysolipids,and glycolipids. The fatty acid composition of the total lipid contentof the soy lecithin used in the present study was analysed and arepresented in FIG. 5 (filled bars). The major fatty acid component islinoleic acid (18:2) constituting 56% of the fatty acids present, otherunsaturated fatty acids are oleic acid (18:1) and linolenic (18:3), andthe unsaturated fatty acids are palmitic (16:0) and stearic acid (18:0).In FIG. 5 data are also presented on the fatty acid composition of thewax ester (open bars) produced from the soy lecithin and ricinoleoylalcohol as described above. By comparing the fatty acid composition ofthe wax ester product with that of the lecithin substrate it is clearthat the unsaturated fatty acids are preferentially converted into thewaxesters whereas the saturated fatty acids are less efficiently usedfor wax ester synthesis. This can partly be explained by the fact thatthese unsaturated fatty acids are preferentially esterified to the sn1position of the lipids and that ATWAX is specific for the transfer offatty acids from the sn-2 position.

The synthesis of different waxesters from soy lecithin was achieved byusing different alcohols as the acyl acceptor and with conditions asfollows; lecithin (2.5 mg),sn1-oleoyl-sn2-[1⁴C]oleoyl-phosphatidylcholine (10 nmol; 5000 dpm/nmol)and 6 mmol of decanol, hexadecanol, 13c-docosanol, hexacosanol orricinoleoyl-alcohol dissolved in chloroform was aliquoted in 12 ml glasstubes and the chloroform was evaporated under a stream of N₂ (g). To thedry lipid substrate 0.5 ml of cell free medium was added and 25 ul 1.0 Mpotassium phosphate, pH 7.2. The reaction mixture was sonicated in awater bath (Branson 2510) for 30 min and was further incubated at 37° C.to a final incubation of 20 hours. The synthesised wax esters wereextracted and quantified as described above. The results are presentedin table 1 and clearly show that apart from hexacosanol all alcoholscould efficiently be used as acyl group acceptors in the synthesis ofwax esters. Hexacosanol is a saturated 26 carbon alcohol with a highmelting point and is therefore badly emulsified at assay conditions usedin the present study and this is suggested to be the main reason to thatonly 5% of added radiolabeled acylgroups were esterified with thehexacosanol. In contrast, approximately 40 to 50 percent of the addedradiolabeled acyl group formed wax esters with the other alcohols tested(table 1). The ricinoleic acid contains a hydroxyl group at position 12in the carbon chain, however it could not act as an acyl acceptor in thecatalyses of wax esters by the ATWAX enzyme. It is therefore concludedthat in the synthesis of waxesters from lecithin and ricinoleoyl alcoholas shown in table 1 the acylgroups derived from the lecithin isexclusively esterified to the hydroxyl group of position 1 and not tothat of position 12 of the ricinoleoyl alcohol.

TABLE 1 Synthesis of wax esters from sn1-oleoyl-sn2-[¹⁴C]oleoyl-phosphatidylcholine and different alcohols (acyl acceptors) in cell freesupernatants. [¹⁴C]-acylgroups in wax esters Acyl acceptor (% of added)Butanol Nd Decanol 42.8 Hexadecanol 44.8 13c-Docosenol 53.9 Hexacosanol5.1 Ricinoleoyl alcohol 51.1 Ricinoleoyl fatty acid nd

Example 6

The ability to catalyse the synthesis of wax ester with the membranebound full length PDAT expressed in Saccharomyces cerevisiae wasexamined. Microsomes were prepared from wild type S. cerevisiae cellsoverexpressing the yeast PDAT gene LRO1 as described in, Dahlqvist etal., 2000 (PNAS 97:6487-6492) and were assayed for wax ester synthesis.The PDAT activity was analysed with the addition of the lipid substratesdissolved in benzene to dry aliquots of lyophilised microsomes(corresponding to 12 nmol of microsomal phosphatidylcholine) (Dahlqvistet al., 2000, PNAS 97:6487-6492). As substrate we used 2.5 nmol ofsn1-oleoyl-sn2-[¹⁴C]oleoyl-phosphatidylcholine (lane 1) together witheither 2.5 nmol ricinoleoyl alcohol (Ric-OH, lane 2) or 13c-docosenol(22:1-OH, lane 3). The enzymatic assay and lipid analysis were performedas described in Dahlqvist et al., 2000 (PNAS 97:6487-6492). It isclearly shown in FIG. 6 that triacylglycerols are synthesised by anacyltransfer of the radiolabeled acyl group of the added phospholipid tothe endogenous diacylglycerols present in the microsomal preparation(FIG. 6, lane 1), as previously reported. However, adding ricinoleoylalcohol or 13c-docosenol (FIG. 6, lane 2 and 3) to the incubation formedno detectable amounts of waxesters. It is therefore concluded that thefull-length membrane associated PDAT encoded by the LRO1 gene do notcatalyse the wax ester synthesis such as presently shown above for themembrane independent acyltransferase, ATWAX. Hence it is here shown thatthe utilisation of different acyl donors and acceptors by an membraneindependent acyltransferase is less limited, as compared toacyltransferases integrated into lipidmembranes via one or severalmembrane-spanning regions, since the accessibility of differentsubstrates are restricted to the vicinity of the localisation of themembrane integrated enzyme.

Example 7

Genes Homologous to the Saccharomyces cerevisiae Gene LRO1.

The yeast PDAT (ScPDAT) amino acid sequence encoded by the LRO1 gene wasused to search the NCBI databases for homologous sequences in plants andmicrobes. In Schizosaccharomyces pombe one gene SpPDAT with stronghomologies to the yeast PDAT gene LRO1 was identified. Four Arabidopsisthaliana genes At6704, At1254, At3027 and At4557 with clear homology toamino acid sequence encoded by the yeast LRO1 gene were identified.Additionally two plant genes Cp6704 and Cp1254 homologies to At6704 andAt1254, respectively, were identified in Crepis palaestina. Thefull-length genes of Cp6704 and Cp1254 were amplified from doublestranded cDNA, synthesised with C. palaestina seed mRNA as template. Thecoding region of the SpPDAT, At6704, At1254, At3027, At4557, Cp6704 andCp1254 nucleic acid sequences are shown in SEQ ID NO:16, 4, 12, 8, 10,18 and 20. The amino acid sequences encoded by SpPDAT, At6704, At1254,At3027, At4557 Cp6704 and Cp1254 sequences, i.w., SEQ ID NO:17, 5, 13,9, 19 and 21, are aligned together with the yeast LRO1 in FIG. 7 usinghierarchical clustering as described in F. Corpet, 1988, Nucl. AcidsRes., 16; 10881-10890. In similarity with the yeast PDAT ScPDAT, aspredicted by the THMM2.0 program (A. Krogh, B. Larsson, G. von Heijne,and E. L. L. Sonnhammer, (2001) Journal of Molecular Biology,305:567-580), all these plant genes contains a single N terminallocalised transmembrane spanning region as marked with gray boxes inFIG. 7. The full length At6704 gene has been shown to encode an enzymewith PDAT activity (Banas et al., 2003 in Advanced Research on PlantLipids 179-182). Any data on the activity associated with the geneproducts of Atl254, At3027 or the At4557 has not yet been published.

Additionally, nucleotide sequences from Aspergillus nidulans andAspergillus fumigatus (SEQ ID NO 14 and 15) were identified. Translatedamino acid sequences from these nucleotide sequences shows stronghomologies with the amino acid sequence of the yeast PDAT (FIG. 8). Insimilarity with the yeast PDAT the Aspergillus sequences contains asingle N-terminal membrane-spanning region, within the first 100 aminoacids, as predicted by the THMM2.0 program.

Example 8

Expression of an Active Membrane Independent Acyltransferase From aNucleotide Sequence, Derived from the Plant Gene At6704.

The yeast PDAT protein sequence was used to search the NCBI databasesfor homologous sequences in Arabidopsis thaliana. One of the identifiedsequences was At6704 encoding a plant PDAT (Banas et al., 2003 inAdvanced Research on Plant Lipids 179-182). A cDNA clone, correspondingto At6704 was ordered from the AIMS database. The clone was sequencedand found to contain an insertion of one base. This extra base wasdeleted through site directed mutagenesis. The At6704 gene encodes aplant-PDAT with a membrane-spanning region from aa 48 to aa 70 aspredicted by the THMM2.0 program (A. Krogh, B. Larsson, G. von Heijne,and E. L. L. Sonnhammer, (2001) Journal of Molecular Biology,305:567-580).

A nucleotide sequence, SEQ ID NO: 6 identical in sequence to the part ofthe plant PDAT gene At6704 encoding the amino acid residues 74 to 671(SEQ ID NO: 7) was amplified by thermo stable Pfu Turbo Polymerase(Stratagene Inc.) from the plasmid template pUS56 containing the fulllength At6704 plant gene. The forward (5′ end) primer used for this PCRreaction was; 5′CCATGGGAATGAATTCGCAATGCCTGCGAGCTTCCCTCAGTATGTA3′ (SEQ IDNO: 28) The oligonucleotide sequence;5′GAATTCGTTAGCGGCCGCCAGCTTCAGGTCAATACGCTCCGACCA3′ (SEQ ID NO: 29) wasused as the reverse (3′ end) primer in the PCR amplification. The aminoacid sequence (SEQ ID NO:7) encoded by the amplified nucleotide sequence(SEQ ID NO:6) lacks the amino acid residues 1 to amino acid 73 includingthe transmembrane region of amino acid residues 48 to 70 present in theplant PDAT as predicted by the THMM2.0 program.

The amplified PCR product above, was purified, digested by EcoRI andNotI, and subcloned between the EcoRI and NotI sites of the Pichiaexpression vector PpicZαA in frame with the N-terminal sequence encodingthe αfactor signalpeptide present in the expression vector and aC-terminal c-myc epitope followed by a polyhistidine tag. The resultantPichia expression vectors are named pHisATWAX-P6. The vector waslinearized using unique SacI restriction site for transformation intoPichia pastoris host strain KM71H. Transformants were cultivated for theexpression of the transformed gene as described in EXAMPLE 2. Todetermine the enzyme activity in the cell free culture medium the cellfree supernatant was assayed for enzyme activity as follows. The cellfree supernatant of the induced cultures of KM71H transformed withpHisATWAX as described in EXAMPLE 2, from 116 hours of growth of Pichiapastoris KM71H transformed with the pHisATWAX-P6 secreting, thepolypeptide HisATWAX-P6 (FIG. 9, lane 1 and 2), the congenic wt strain(FIG. 9, lane 3 and 4) and KM71H expressing the polypeptide ATWAX wereassayed for acyl transferase activity.

.Lipid substrate, sn1-palmitoylsn2-[¹⁴C]linoleoyl-phosphatidylethanolamine (5 nmol; 5000 dpm/nmol) anddioleoylglycerol (2.5 nmol) dissolved in chloroform was aliquoted in 1.5ml tubes and the chloroform was evaporated under a stream of N₂ (g).After addition of 20 ul 0.25 M potassium phosphate, pH 7.2, the mixturewas violently agitated and 80 ul of cell free supernatant was added andincubated at 30° C. for 90 min. Lipids were extracted from the reactionmixture into chloroform (Bligh, E. G. and Dyer, W. J (1959) Can. J.Biochem. Physiol. 37, 911-917) and separated by TLC on silica gel 60plates (200×200 mm) in chloroform/methanol/acetic acid/water(85:15:10:3.5) migrating 90 mm using an automatic developing chamber(Camag). The plate was dried and redeveloped in hexane/diethylether/acetic acid (80:20:1) with a solvent migration of 180 mm. Theradioactive lipids were visualized and quantified on the plates byelectronic autoradiography (Instant Imager; Packard). As a control, theenzyme activity in the cell free supernatant of the wild type hoststrain culture was analysed. As shown in FIG. 9, radiolabeledtriacylglycerol (TAG) is formed, from the added lipid substrate,sn1-palmitoyl sn2-[¹⁴C]linoleoyl-phosphatidylethanolamine anddioleoylglycerol, in cell free extract of Pichia strain transformed withpHisATWAX-P6. This demonstrates that the truncated membrane independentform of the plant PDAT, referred to as a membrane independentacyltransferase that we have named HisATWAX-P6, is able of catalysingthe formation of TAG by an acyltransfer from phosphatidylethanolamine todiacylglycerol (DAG).

1. An isolated polypeptide obtained from a polypeptide comprising theamino acid sequence set forth in SEQ ID NO:3, wherein said polypeptidecomprising the amino acid sequence set forth in SEQ ID NO:3 is anintegral membrane protein comprising a membrane spanning region, whereinamino acid residues 1 to 97 have been deleted from the N-terminus ascompared to the amino acid sequence set forth in SEQ ID NO:3, andwherein the polypeptide has membrane independent acyltransferaseactivity.
 2. The polypeptide according to claim 1 ,wherein thepolypeptide is an acyltransferase active at a pH range of from about 4to about 10 and stable at a temperature below 60° C.
 3. The polypeptideaccording to claim 2, wherein the polypeptide is an acyltransferaseactive at a pH of 7.2 at a temperature of about 30° C.
 4. Thepolypeptide according to claim 1, wherein the polypeptide is immobilizedto a carrier.
 5. The polypeptide according to claim 1, wherein thepolypeptide is lyophilized and/or freeze-dried.
 6. A kit comprising thepolypeptide according to claim 1 and a stabilizer.
 7. The kit accordingto claim 6, wherein the polypeptide is in a lyophilized form orfreeze-dried.
 8. A kit according to claim 6, wherein the polypeptide isimmobilized to a carrier.
 9. A detergent composition comprising thepolypeptide of claim
 1. 10. A food composition comprising thepolypeptide of claim
 1. 11. A method for removal of a phospholipid fromvegetable oil, comprising contacting the vegetable oil with thepolypeptide of claim 1, wherein fatty acids are transferred from saidphospholipid to an acceptor molecule present in said vegetable oil. 12.A method according to claim 11, wherein said phospholipid is lecithin,wherein said lecithin is converted to lysolecithin, and wherein saidlysolecithin is removed from the oil into a water phase.
 13. Apolypeptide according to claim 1, wherein said membrane independentacyltransferase activity comprises ester synthesis.
 14. A polypeptideaccording to claim 13, wherein said ester synthesis comprises wax estersynthesis.
 15. A method for preparing a baked product from a doughcomprising converting polar lipids in a flour into lysolipids, saidmethod comprising contacting a dough comprising said flour with apolypeptide according to claim 1 to said dough, wherein polar lipids insaid flour are enzymatically converted into lysolipids by saidpolypeptide, thereby preparing the baked product.
 16. A method forconverting lecithin or phospholipids to lysolecithin orlysophospholipids in a food material, comprising contacting said foodmaterial with a polypeptide according to claim
 1. 17. A method accordingto claim 16, wherein said food material is selected from the groupconsisting of milk, flour, eggs, soy protein, and cocoa.